Abstract
Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.
Highlights
Cyclic AMP allosterically modulates GNAT-like acyltransferases (KAT) in mycobacteria
Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases
An ␣-helix (␣10 or C-terminal helix), a unique extension seen in these acyltransferases, that spatially connects the AT domain with the cAMP binding site in the cyclic nucleotide binding (CNB) domain (Figs. 1A and 2)
Summary
Cyclic AMP allosterically modulates GNAT-like acyltransferases (KAT) in mycobacteria. Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. We provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. We have recently reported that cAMP can regulate the acylation of lysine residues in mycobacterial proteins by binding to unique cAMP-regulated acyltransferases (6, 7) We called these proteins lysine (K) acyltransferases from Mycobacterium smegmatis (KATms)[3] and Mycobacterium tuberculosis (KATmt). The structures we describe here, along with confirmatory mutational analysis, identify distinct features that account for the divergent activities of KATms and KATmt in the absence of cAMP.
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